ABSTRACT
Objective:To investigate the clinical and biological characteristics of relapsed childhood low-risk acute B lymphoblastic leukemia (B-ALL).Methods:The clinical and laboratory data of 34 children who admitted in Beijing Boren Hospital from July 2017 to July 2018 were retrospectively analyzed, and 127-339 mutations of hematological malignancy related genes were analyzed.Results:The median time from the diagnosis to the recurrence was 871 d (87-1 446 d). The recurrence at early stage and late stage had 26 cases (76%) and 8 cases (24%), respectively. The recurrence before maintenance treatment, during maintenance therapy and after withdrawal of chemotherapy had 3 cases (9%), 12 cases (35%) and 19 cases (56%) (13 cases relapsed within 1 year after withdrawal, 6 cases relapsed after withdrawal 1-2 years and no one relapsed after withdrawal 2 years). The sites of recurrence included bone marrow alone accounting for 26 cases (76%), both intramedullary and extramedullary disease (EMD) accounting for 6 cases (18%), EMD alone accounting for 2 cases (6%). Flow cytometry showed that 9 patients presented minimal residual disease (MRD)-positive (6 cases with one positive, 2 cases with twice positive and 1 case with 3 times positive), including 8 cases occurred at early stage and 1 case occurred at late stage; and the level of MRD was 0.02%-3.82%. Complex chromosomal karyotype appeared in 6 relapsed children with normal or hyperdiploid karyotype at first diagnosis. Hematological malignancy related gene mutation detection was made in 28 cases, and the results showed that each patient had at least one gene mutation, and 2 or more gene mutations were detected in 25 cases (89%). The high frequency of gene mutations were as follows: CREBBP (7 cases, 25%), NRAS (7 cases, 25%), KRAS(7 cases, 25%), TP53 (4 cases, 14%), and NT5C2 (4 cases, 14%).Conclusions:The recurrence of childhood low-risk B-ALL occurs mostly in the maintenance treatment or in two years of withdrawal of chemotherapy. Positive MRD after complete remission is likely to show the risk of early recurrence. The gene mutations after the poor prognosis in cancer cells may be related to the recurrence of childhood low-risk B-ALL, and the common gene mutations include CREBBP, RAS signaling pathways genes and TP53, NT5C2.
ABSTRACT
Objective To investigate the association of genetic polymorphisms of ATIC and GSTP1 with plasma concentrations and adverse reactions of high-dose methotrexate( HD-MTX)in children with acute lymphoblastic leukemia (ALL). Methods A total of 70 peripheral blood samples were obtained from ALL children for extraction of genome DNA.The gene polymorphisms of ATIC T26293C and GSTP1 A313G locus were examined by using PCR and direct sequencing.Enzyme multiplied immunoassay technique(EMIT)was employed to determine the plasma concentration of MTX in 48 h.Clinical data of patients were collected during HD-MTX chemotherapy,and the adverse reactions were statistically analyzed.The associations of ATIC and GSTP1 genotypes with MTX plasma concentration and adverse reactions were investigated. Results There were genetic polymorphisms at the SNP of ATIC T26293C and GSTP1 A313G.At the SNP of ATIC T26293C,the percentages of TT, CT and CC genotypes in ALL children were 4.35%,39.13% and 56.52%,respectively,and the frequencies of T and C alleles were 23.91% and 76.09%.At the SNP of GSTP1 A313G,the percentages of AA,GA and GG genotype were 68.57%,28.57%and 2.86%,respectively,in ALL children. The frequencies of A and G alleles were 82. 86% and 17. 14%,respectively. No statistically significant difference was found in the ratio of blood MTX concentration to MTX dose at 48 h between children with different genotypes(P>0.05).In the GSTP1 A313G site,genotypes that induced the gastrointestinal reactions in the order from low to high were AA,GA,GG,and there was a significant association between gene polymorphism and gastrointestinal side effects(P<0.05).In the GSTP1 A313G site,genotypes that induced myelosuppression in the order of low to high were GG,AA, GA,and a significant association was noted between gene polymorphism and myelosuppression(P<0.05). Conclusion There are significant associations between GSTP1 A313G polymorphism and gastrointestinal side effects or myelosuppression after HD-MTX chemotherapy in ALL children.
ABSTRACT
Objective To explore the role of murine double minute 2 (MDM2) in apoptosis of acute lymphoblastic leukemia EU-4 cells induced by berberine.Methods EU-4 cells at logarithm growth phase were chosen to carry out the experiments.EU-4 cells were incubated with different doses of berberine for 72 hours,and the final doses of berberine were 0,1,10 and 100 μmoL/L,respectively.Then the apoptosis rate of EU-4 cells were detected by flow cytometry,and the expression levels of MDM2 protein were tested by Western blot.The levels of MDM2 gene were examined by means of real-time PCR at 0,24,48 and 72 hours after incubation of EU-4 cells with 100 μmol/L berberine,respectively.EU-4 cells in MDM2 small interfering RNA(siRNA) group were transfected with 150 nmol/L MDM2 siRNA,and the control siRNA group were transfected with 150 nmol/L control siRNA.The apoptosis rates of EU-4 cells were detected 48 hours after transfection by flow cytometry.Results The apoptosis rates of EU-4 cells were increased in a dosedependent manner,and the highest apoptosis rate was up to(60.13 ± 4.21)％,which was incubated with 100 μmol/L berberine.Significant differences were found in all groups (F =280.56,P ＜ 0.05).Berberine treatment suppressed the protein expression of MDM2 in a dose-dependent manner(F =73.82,P ＜ 0.01).The mRNA expression of MDM2 was inhibited in a time-dependent manner by 100 μmol/L berberine(F =45.37,P ＜0.01).The apoptosis rates of control siRNA group and MDM2 siRNA group were(11.09 ± 1.63) ％ and (29.84 ± 1.75) ％,respectively.Significant difference was found between the 2 groups (t =-13.57,P ＜ 0.01).Conclusions Berberine can inhibit the expression of MDM2 in a dose-and time-dependent manner,which may be one of the mechanisms of apoptosis induced by berberine in leukemia cells.
ABSTRACT
Objective To analyze the clinical features of pediatric patients with acute lymphoblastic leukemia(ALL) with bcr-abl fusion gene transcript,and discuss the treatment,prognosis factors of this kind of ALL.Methods Clinical features,treatment and prognosis were studied retrospectively in 7 bcr-abl fusion gene positive ALL patients.bcr-abl fusion gene was detected by reverse transcription polymerase chain reaction (RT-PCR).Results The average age of the 7 patients was 8 years and 1 month old.All of them were common B-immunology ALL.The rate of complete response was 50 ％ after 33 days' treatment.Conclusions The incidence rate of bcr-abl fusion gene positive ALL in pediatric is low.This type of ALL has poor remission,high relapse rate and poor prognosis.
ABSTRACT
Objective To study the molecular mechanism of berberine induced apoptosis in chemoresistant EU-4 acute lymphocytic leukemia cells.Methods EU-4 cells were treated with 0,10 and 100 μmol/L berberine for 72 h.The apoptosis induced by berberine was detected by flow cytometry.The expression of Caspase-3,PARP and X-linked inhibitor of apoptosis protein (XIAP) were determined by Western bolt assay.Caspase-3 activity was measured using microplate reader.After transfected with XIAP siRNA,the apoptosis was detected by flow cytometry.Results After treated with 0,10 and 100 μmol/L berberine for 72 h,the apoptosis rates of EU-4 cells were (9.08±1.20) ％,(22.36±2.16) ％ and (59.81±4.17) ％,respectively.Berberine induced potent apoptosis in a dose-dependent manner.The apoptosis involved activation of Caspase-3.The Caspase-3 activities were 1.70±0.25,1.92±0.10 and 2.89±0.25,respectively.Berberine inhibited XIAP expression in a dose-and time-dependent manner (P ＜ 0.05).Down-regulation of XIAP by siRNA increased apoptosis of EU-4 cells.The apoptosis rates were (9.23±1.66) ％ and (22.15±0.63) ％.Conclusion Berberine could induce apoptosis of EU-4 cells,and inhibition of XIAP leading to Caspase-3 activation is responsible for the apoptotic effect on EU-4 cells.
ABSTRACT
The expression of silience of death domains (SODD) and its clinical significance and relationship with phospho-NF-κB-p65 proteins in bone marrow cells of childhood acute lymphoblastic leukaemia (ALL) were explored, and the expression of SODD and phospho-NF-κB-p65 in Jurkat cells treated with chemotherapeutic drugs was detected in order to find a new chemotherapeutic target. The expression of SODD and phospho-NF-κB-p65 proteins in bone marrow cells was detected by immunohistochemistry in 25 children with ALL. The apoptosis rate was measured by Annexin-V-Fluorescence/PI double-labeling flow cytometry and the expression of SODD and phospho-NF-κB-p65 proteins determined by Western blotting in the Jurkat cells. It was found that the expression of SODD and active P65 in ALL was significantly higher than that in normal control group (P＜0.05). The expression of the SODD and phospho-NF-κB-p65 proteins in the high-risk (HR) group was significantly higher than that in the standard-risk (SR) group (P＜0.05). The Pearson rank correlation analysis revealed that there was a positive correlation between SODD and phospho-NF-κB-p65 expression (P＜0.01, r=0.69). VCR could effectively induce the apoptosis of Jurkat cells, and down-regulate the expression of SODD and phospho-NF-κB-p65 proteins in a time-dependent manner, but DNR could not down-regulate the expression of SODD effectively. It was concluded that SODD may be closely related to the clinical classification and prognosis of ALL in children. The expression of SODD and phospho-NF-κB-p65 had a definite synergistic relationship with the onset and development of ALL. VCR could down-regulate the expression of SODD and inhibit the NF-κB activation, which could recover the sensibility of apoptosis in leukemic cells.
ABSTRACT
The expression of silence of death domains (SODD) and its clinical significance and relationship with phospho-NF-kappaB-p65 proteins in bone marrow cells of childhood acute lymphoblastic leukaemia (ALL) were explored, and the expression of SODD and phospho-NF-kappaB-p65 in Jurkat cells treated with chemotherapeutic drugs was detected in order to find a new chemotherapeutic target. The expression of SODD and phospho-NF-kappaB-p65 proteins in bone marrow cells was detected by immunohistochemistry in 25 children with ALL. The apoptosis rate was measured by Annexin-V-Fluorescence/PI double-labeling flow cytometry and the expression of SODD and phospho-NF-kappaB-p65 proteins determined by Western blotting in the Jurkat cells. It was found that the expression of SODD and active P65 in ALL was significantly higher than that in normal control group (P<0.05). The expression of the SODD and phospho-NF-kappaB-p65 proteins in the high-risk (HR) group was significantly higher than that in the standard-risk (SR) group (P<0.05). The Pearson rank correlation analysis revealed that there was a positive correlation between SODD and phospho-NF-kappaB-p65 expression (P<0.01, r=0.69). VCR could effectively induce the apoptosis of Jurkat cells, and down-regulate the expression of SODD and phospho-NF-kappaB-p65 proteins in a time-dependent manner, but DNR could not down-regulate the expression of SODD effectively. It was concluded that SODD may be closely related to the clinical classification and prognosis of ALL in children. The expression of SODD and phospho-NF-kappaB-p65 had a definite synergistic relationship with the onset and development of ALL. VCR could down-regulate the expression of SODD and inhibit the NF-kappaB activation, which could recover the sensibility of apoptosis in leukemic cells.
ABSTRACT
The threshold of cyclin E expression at G1/S boundary is a characteristic feature of cell cycle progressing. In this study, we tried to develop a quantitative approach to analyze cyclin E threshold by multiparameter flow cytometry. The expression of cyclin E in exponentially growing MOLT-4 cells was detected under different photomultiplier tube (PMT) voltages by cyclin E/DNA multiparameter flow cytometry. Additionally, cyclin E was detected in cells which were treated with caffeine and cycloheximide (CHX) under the same PMT voltage. Moreover, the expression of cyclin E in MOLT-4 cells was compared with that in JURKAT cells. Cyclin E threshold was quantified by formula B2/AxC (A, B, C indicates the minimum, threshold, and maximum of cyclin E fluorescence intensity, respectively). Results showed that in MOLT-4 cells, cyclin E threshold calculated by formula B2/AxC was invariable under different PMT settings. It was decreased in cells treated with caffeine and remained changeless in cells treated with cycloheximide. Cyclin E threshold in JURKAT cells was much lower than that in MOLT-4 cells. It was suggested that Formula B2/AxC we firstly set up could be used to analyze cyclin E expression threshold quantitatively.
Subject(s)
Caffeine/pharmacology , Cell Cycle/physiology , Cell Line, Tumor , Cyclin E/analysis , Cycloheximide/pharmacology , DNA, Neoplasm/analysis , Flow Cytometry/methods , Jurkat Cells , Leukemia, Lymphoid/pathologyABSTRACT
Summary: The threshold of cyclin E expression at G1/S boundary is a characteristic feature of cell cycle progressing. In this study, we tried to develop a quantitative approach to analyze cyclin E threshold by multiparameter flow cytometry. The expression of cyclin E in exponentially growing MOLT-4 cells was detected under different photomultiplier tube (PMT) voltages by cyclin E/DNA multiparameter flow cytometry. Additionally, cyclin E was detected in cells which were treated with caffeine and cycloheximide (CHX) under the same PMT voltage. Moreover, the expression of cyclin E in MOLT-4 cells was compared with that in JURKAT cells. Cyclin E threshold was quantified by formula B2/A×C (A, B, C indicates the minimum, threshold, and maximum of cyclin E fluorescence intensity, respectively). Results showed that in MOLT-4 cells, cyclin E threshold calculated by formula B2/A×C was invariable under different PMT settings. It was decreased in cells treated with caffeine and remained changeless in cells treated with cycloheximide. Cyclin E threshold in JURKAT cells was much lower than that in MOLT-4 cells. It was suggested that Formula B2/A×C we firstly set up could be used to analyze cyclin E expression threshold quantitatively.